Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans

Abstract

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af-Tth) was determined by X-ray crystallography to a resolution of 1.95 Å. Af-Tth is a homodimer, and its monomer structure exhibits an eight-bladed β-propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the β-propeller region. We observed unexplained electron densities in this cavity of the substrate-soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site-specific Af-Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af-Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step.

Keywords: Acidithiobacillus ferrooxidans; hydrolase; protein tertiary structure; site-directed mutagenesis; sulfur oxidation; tetrathionic acid.

FIGURE 1

Overall structure of recombinant tetrathionate hydrolase from A. ferrooxidans. Monomer (a) and dimer (b) structures. Each monomer of chains B and D in the dimer is colored yellow and magenta (β‐strands), respectively, red and cyan (α‐ and 3₁₀ helices), respectively, and green and wheat (loops), respectively. Monomer (a) is the view from the right side of (b). The monomer structure exhibits an eight‐bladed (I–VIII) β‐propeller motif. L1 and L2 are the insertion loops of the eight‐bladed β‐propeller motif

FIGURE 2

Active site cavity of tetrathionate hydrolase from A. ferrooxidans. (a) The side view of the wild‐type monomer (chain F). (b) A close‐up view of the rectangular box in (a). Cavities were displayed using the Cavities & Pockets (Culled) command of PyMOL. (c) Electron densities in the cavity of the substrate‐soaked structure (chain C). Side chains of residues lining the active site cavity are indicated as a stick model. The water molecules and the three, surrounding methionine residues (M172, M238, and M279) in the cavity are marked with red and black arrows, respectively. In (c), methionines, arginines, aspartic acid, hydrophobic residues (Val, Ala, Phe, Ile), and other residues (Ser, Thr, Asn, Gln) are colored orange, cyan, magenta, green, and wheat, respectively. The green and yellow contour shows the F _o − F _c omit map (3.0σ, λ = 1.0 Å) and anomalous difference map (3.0σ, λ = 1.9 Å), respectively

FIGURE 3

Activity measurement. The activities of wild‐type and D325N tetrathionate hydrolase from Acidithiobacillus ferrooxidans are indicated by closed and open circles, respectively. The activities were determined by monitoring the tetrathionate concentration in each reaction mixture

FIGURE 4

Electrostatic surface potentials of tetrathionate hydrolase dimers at pH 2.0. Van der Waals surfaces of the tetrathionate hydrolase from A. ferrooxidans (Af‐Tth, upper figure) and tetrathionate hydrolase from Ad. ambivalens (Ad‐TTH1) homology models (lower figure) are colored according to the local electrostatic potential as calculated using APBS ⁴¹ from −4kT (red) to +4kT (blue)

FIGURE 5

Proposed catalytic mechanism of tetrathionate hydrolysis by tetrathionate hydrolase from A. ferrooxidans. Asp325 works as a general acid under neutral or acidic conditions